We compared effects of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-<em>121</em> (VEGF<em>121</em>) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-165 (VEGF165) on generation of NO in HUVEC and the involvement of NO in VEGF<em>121</em>- and VEGF165-induced angiogenesis. VEGF stimulated synthesis of NO within seconds, reaching peak concentrations of 450 +/- 25 and 180 +/- 15 nmol/l for VEGF<em>121</em>, and VEGF165, respectively. The VEGF<em>121</em> increased NO production for about 40 s while VEGF165-stimulated NO release lasted only for about 20 s. Accordingly, cGMP elevation was stronger in VEGF<em>121</em>- than in VEGF165-treated cells. The VEGF<em>121</em> was a very weak mitogen but strong chemoattractant for HUVEC, whereas VEGF165 potently induced both cell proliferation and migration. NO appeared to be involved in the <em>endothelial</em> migration and morphogenesis but not in the proliferation. NO was also a permissive molecule for VEGF<em>121</em>- but not for VEGF165-induced capillary sprouting in spheroid culture. In conclusion, VEGF<em>121</em> is a stronger stimulator of <em>endothelial</em> nitric oxide synthase (eNOS) activity, and angiogenic potential of VEGF<em>121</em> is more reliant on NO contribution.

OBJECTIVE

To investigate cytokine- and oxidation-related genes for preeclampsia using DNA microarray analysis.

METHODS

Placentas were collected from 13 normal pregnancies and 13 patients with preeclampsia. Gene expression was studied using DNA microarray. Among significantly expressed genes, we focused on genes associated with cytokines and oxidation, and the results were confirmed using quantitative real time-polymerase chain reaction (QRT-PCR).

RESULTS

415 genes out of 30,940 genes were altered by>> or =2-fold in the microarray analysis. <em>121</em> up-regulated genes and 294 down-regulated genes were found to be in preeclamptic placenta. Six cytokine-related genes and 5 oxidation-related genes were found from among the <em>121</em> up-regulated genes. The cytokine-related genes studied included oncostatin M (OSM), fms-related tyrosine kinase (FLT1) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGFA), and the oxidation-related genes studied included spermine oxidase (SMOX), l cytochrome P450, family 26, subfamily A, polypeptide 1 (CYP26A1), acetate dehydrogenase A (LDHA). These six genes were also significantly higher in placentas from patients with preeclampsia than in those from women with normal pregnancies. The placental tissue of patients with preeclampsia showed significantly higher mRNA expression of these six genes than the normal group, using QRT-PCR.

CONCLUSIONS

DNA microarray analysis is one of the great methods for simultaneously detecting the functionally associated genes of preeclampsia. The cytokine-related genes such as OSM, FLT1 and VEGFA, and the oxidation-related genes such as LDHA, CYP26A1 and SMOX might prove to be the starting point in the elucidation of the pathogenesis of preeclampsia.

Tumor angiogenesis is a critical step for the <em>growth</em> and metastasis of solid tumors. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a specific and potent angiogenic <em>factor</em> and contributes to the development of solid tumors by promoting tumor angiogenesis. Therefore, it is a prime therapeutic target for the development of antagonists for treatment of cancer. We identified from peptide libraries arginine-rich hexapeptides that inhibit the interaction of VEGF(165) with VEGF receptor (IC(50) = 2-4 micrometer). They have no effect on binding of basic fibroblast <em>growth</em> <em>factor</em> to cellular receptor. The hexapeptides inhibit the proliferation of human umbilical vein <em>endothelial</em> cells induced by VEGF(165) without toxicity. The peptides bind to VEGF and inhibit binding of both VEGF(165) and VEGF(<em>121</em>), suggesting that the peptides interact with the main body of VEGF but not the heparin-binding domain that is absent in VEGF(<em>121</em>). The identified peptides block the angiogenesis induced by VEGF(165) in vivo in the chick chorioallantoic membrane and the rabbit cornea. Furthermore, one of the hexapeptides, RRKRRR, blocks the <em>growth</em> and metastasis of VEGF-secreting HM7 human colon carcinoma cells in nude mice. Based on our results, the arginine-rich hexapeptides may be effective for the treatment of various human tumors and other angiogenesis-dependent diseases that are related to the action of VEGF and could also serve as leads for development of more effective drugs.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) was discovered 10 years ago as a <em>growth</em> <em>factor</em> that can regulate angiogenesis and in addition the permeability of blood vessels. Numerous studies have revealed that it is essential for normal embryonic development and that it plays a major role in physiological and pathological events of angiogenesis in adults. It is unique in that its expression is regulated directly by hypoxia. These properties are now being exploited in attempts aimed at the induction of new blood vessels in pathological situations such as ischemic heart disease. Five VEGF forms of <em>121</em> to 206 aminoacids are produced from a single gene by alternative splicing. Cells expressing VEGF usually express several forms simultaneously. VEGF<em>121</em> does not contain exons 6 and 7 of the gene and consequently lacks a heparin binding ability. However, this form is fully active as an inducer of angiogenesis, and as a blood vessel permeabilizing agent. Exon 6 and 7 contain 2 independent heparin binding domains. The VEGF form containing exon 7 (VEGF165) and the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> form containing exon 6 (VEGF145) display similar biological potencies raising the question of why so many VEGF forms are required. It was found that VEGF<em>121</em> diffuses better because it does not bind to heparan-sulfate proteoglycans. In contrast, VEGF145 binds to extracellular matrix and is released from it slowly. When the receptor binding properties of VEGF<em>121</em> and VEGF165 were compared it was found that VEGF165 binds to a class of VEGF receptors that is not recognized by VEGF<em>121</em>. These receptors are encoded by the neuropilin-1 gene, and we have recently found that the related neuropilin-2 gene also encodes a VEGF165 receptor. We have recently found evidence indicating the neuropilins form complexes with another VEGF receptor, VEGFR-1. However, the biological function of this complex remains to be elucidated.

Experiments were designed to investigate the expression and regulation of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) in the primate corpus luteum (CL) throughout the luteal life span in the natural menstrual cycle. Corpora lutea were collected during the early (ECL; Days 3-5 post-LH surge), mid (MCL; Day 6-8 post-LH surge), mid-late (MLCL; Days 10-12 post-LH surge), late (LCL; Days 14-16 post-LH surge), and very late (Days 17- 18 post-LH surge) luteal phase. Specific primers were designed to amplify mRNAs encoding VEGF isoforms 206, 189, 183, 165, 145, and <em>121</em>. Only two cDNA products were obtained by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends; cloning and sequencing confirmed their 98% homology to the corresponding human VEGF 165 and <em>121</em> sequences. Semiquantitative RT-PCR assays indicated that VEGF 165 mRNA levels increased (P < 0.05) from ECL to MLCL but then declined (P < 0.05) by LCL. Although VEGF <em>121</em> mRNA levels were limited in ECL, they increased significantly in MCL (P < 0.05). Levels of VEGF protein, as measured by Western blot analysis, were two- to fourfold higher for VEGF 165 versus VEGF <em>121</em>. Also, VEGF 165 levels were higher (P < 0.05) in ECL and MCL compared to those at later stages. During 2-day culture, preparations of dispersed luteal cells secreted VEGF into the media; the highest levels were observed in ECL and declined (P < 0.05) by LCL. Regardless of luteal stage, hypoxic conditions increased (P < 0.05) VEGF levels, whereas LH exposure increased (P < 0.05) progesterone, but not VEGF, in the media. These results are consistent with a dynamic, local regulation of VEGF production during the life span of the primate CL that is not directly controlled by LH.

To assess the hypothesis that angiogenic gene therapy with the genomic form of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) expressing the three major isoforms could be more potent than a vector expressing a single isoform, we designed an adenovirus vector (AdVEGF-All) expressing a VEGF cDNA/genomic hybrid gene. AdVEGF-All expressed all three major isoforms (<em>121</em>, 165, 189) in a 2:2:1 ratio. AdVEGF-All was 100-fold more potent than cDNA vectors expressing VEGF <em>121</em>, 165, or 189 in restoring blood flow to the ischemic mouse hind limb. Interestingly, a mixture of Ad vectors individually expressing the VEGF <em>121</em>, 165, and 189 cDNAs was equipotent to an equivalent dose of AdVEGF-All. Thus, a mixture of VEGF isoforms provides a more potent angiogenic response than a single isoform, suggesting that the individual isoforms function synergistically, an observation with important implications for gene and recombinant protein therapy.

OBJECTIVE

To determine monitoring and treatment patterns and vision outcomes in real-world patients initiating anti-vascular endothelial growth factor (anti-VEGF) therapy for diabetic macular edema (DME).

METHODS

Retrospective interventional cohort study.

METHODS

Setting: Electronic medical record analysis of Geisinger Health System data.

METHODS

A total of 110 patients (121 study eyes) initiating intravitreal ranibizumab or bevacizumab for DME during January 2007‒May 2012, with baseline corrected visual acuity of 20/40 to 20/320, and ≥1 ophthalmologist visit during follow-up.

METHODS

Intravitreal injections per study eye during the first 12 months; corrected visual acuity, change in corrected visual acuity from baseline, proportions of eyes with ≥10 or ≥15 approximate Early Treatment Diabetic Retinopathy Study letter gain/loss at 12 months; number of ophthalmologist visits.

RESULTS

Over 12 months, mean number of ophthalmologist visits was 9.2; mean number of intravitreal injections was 3.1 (range, 1-12), with most eyes (68.6%) receiving ≤3 injections. At 12 months, mean corrected visual acuity change was +4.7 letters (mean 56.9 letters at baseline); proportions of eyes gaining ≥10 or ≥15 letters were 31.4% and 24.0%, respectively; proportions of eyes losing ≥10 or ≥15 letters were 10.8% and 8.3%, respectively. Eyes receiving adjunctive laser during the first 6 months (n = 33) showed similar change in corrected visual acuity to non-laser-treated eyes (n = 88) (+3.1 vs +5.3 letters at 12 months).

CONCLUSIONS

DME patients receiving anti-VEGF therapy in clinical practice undergo less frequent monitoring and intravitreal injections, and achieve inferior vision outcomes to patients in landmark clinical trials.

A catalogue of proteins in the human vitreous humor may contribute to elucidating the pathogenesis of various diseases in ophthalmology. To improve the recovery of proteins in vitreous, we applied one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-PAGE). Proteins were extracted from unstained gel strips and digested in gel with trypsin and the peptides were analyzed by capillary-column reversed-phase high-performance liquid chromatography coupled with electrospray ionization-ion trap-mass spectrometry. From a patient with diabetic retinopathy, 84 different proteins were identified. Most of the proteins which we identified in vitreous previously using 2D-PAGE were also identified in the present study. In total, we identified <em>121</em> different proteins including five proteins seen at the genomic level only. Four angiogenic <em>factors</em>, insulin-like <em>growth</em> <em>factor</em>, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>, fibroblast <em>growth</em> <em>factor</em>, and placental <em>endothelial</em> cell <em>growth</em> <em>factor</em>, and three anti-angiogenic <em>factors</em>, pigment epithelium-derived <em>factor</em>, endostatin, and thrombospondin, were found, and this may contribute to elucidating the pathological changes in the concentration and the modified structures of these proteins, in diseases of the retina, especially, diabetic retinopathy.

Plants and mammals are separated by a huge evolutionary distance. Consequently, biotechnology and genetics have traditionally been divided into 'green' and 'red'. Here, we provide comprehensive evidence that key components of the mammalian transcription, translation and secretion machineries are functional in the model plant Physcomitrella patens. Cross-kingdom compatibility of different expression modalities originally designed for mammalian cells, such as native and synthetic promoters and polyadenylation sites, viral and cellular internal ribosome entry sites, secretion signal peptides and secreted product proteins, and synthetic transactivators and transrepressors, was established. This mammalian expression portfolio enabled constitutive, conditional and autoregulated expression of different product genes in a multicistronic expression format, optionally adjusted by various trigger molecules, such as butyrolactones, macrolide antibiotics and ethanol. Capitalizing on a cross-kingdom-compatible expression platform, we pioneered a prototype biopharmaceutical manufacturing scenario using microencapsulated transgenic P. patens protoplasts cultivated in a Wave Bioreactor. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF(<em>121</em>)) titres matched those typically achieved by standard protonema populations grown in stirred-tank bioreactors. The full compatibility of mammalian expression systems in P. patens further promotes the use of moss as a cost-effective alternative for the manufacture of complex biopharmaceuticals, and as a valuable host system to advance synthetic biology in plants.

The anticoagulant serpin antithrombin acquires a potent antiangiogenic activity upon undergoing conformational alterations to cleaved or latent forms. Here we show that antithrombin antiangiogenic activity is mediated at least in part through the ability of the conformationally altered serpin to block the proangiogenic <em>growth</em> <em>factors</em> fibroblast <em>growth</em> <em>factor</em> (FGF)-2 and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) from forming signaling competent ternary complexes with their protein receptors and heparan sulfate co-receptors on <em>endothelial</em> cells. Cleaved and latent but not native forms of antithrombin blocked the formation of FGF-2-FGF receptor-1 ectodomain-heparin ternary complexes, and the dimerization of these complexes in solution and similarly inhibited the formation of FGF-2-heparin binary complexes and their dimerization. Only antiangiogenic forms of antithrombin likewise inhibited (125)I-FGF-2 binding to its low affinity heparan sulfate co-receptor and blocked FGF receptor-1 autophosphorylation and p42/44 MAP kinase phosphorylation in cultured human umbilical vein <em>endothelial</em> cells (HUVECs). Moreover, treatment of HUVECs with heparinase III to specifically eliminate the FGF-2 heparan sulfate co-receptor suppressed the ability of antiangiogenic antithrombin to inhibit <em>growth</em> <em>factor</em>-stimulated proliferation. Antiangiogenic antithrombin inhibited full-length VEGF(165) stimulation of HUVEC proliferation but did not affect the stimulation of cells by the heparin-binding domain-deleted VEGF(<em>121</em>). Taken together, these results demonstrate that antiangiogenic forms of antithrombin block the proangiogenic effects of FGF-2 and VEGF on <em>endothelial</em> cells by competing with the <em>growth</em> <em>factors</em> for binding the heparan sulfate co-receptor, which mediates <em>growth</em> <em>factor</em>-receptor interactions. Moreover, the inability of native antithrombin to bind this co-receptor implies that native and conformationally altered forms of antithrombin differentially bind proangiogenic heparan sulfate domains.

OBJECTIVE

To investigate whether vector-based vascular endothelial growth factor 165 (VEGF)(165) targeted siRNA expression system (pSilencer(siVEGF)) could inhibit VEGF(165) expression in vitro and suppresses retinal neovascularization in the murine model of oxygen-induced retinopathy.

METHODS

pSilencer(siVEGF), from which siRNA targeting VEGF(165) could be generated, was constructed and transfected to human umbilical vein endothelial cells. Then the level of VEGF isoforms in cultured cells was measured by RT-PCR and ELISA. Intravitreal injection of pSilencer(siVEGF) was performed in mice with ischemic retinopathy. Retinal neovascularization was evaluated by angiography using fluorescein-labeled dextran and quantitated histologically. The levels of VEGF(164), which is equivalent to human VEGF(165) in murine retinas were determined by RT-PCR and western immunoblotting.

RESULTS

Expression of VEGF(165) in cultured cells was greatly curtailed by pSilencer(siVEGF) under both normoxia and hypoxia conditions. However, the other isoforms, VEGF(189) and VEGF(121), were expressed to a similar degree regardless of whether pSilencer(siVEGF) was administered. Based on angiography and histological analysis, retinal neovascularization in the eyes treated with pSilencer(siVEGF) were significantly reduced compared to the control eyes. Furthermore, the VEGF(164) levels in the murine retinas were suppressed by pSilencer(siVEGF).

CONCLUSIONS

Retinal neovascularization in the murine model was significantly attenuated by pSilencer(siVEGF) through decreasing VEGF(164) levels in the retinas. pSilencer(siVEGF) seems to be a potential therapeutic tool for ischemic-induced retinal diseases.

Angiogenesis is induced by soluble <em>factors</em> such as <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) released from tumor cells in hypoxia. It enhances solid tumor <em>growth</em> and provides an ability to establish metastasis at peripheral sites by tumor cell migration. Thymosin beta-4 (TB4) is an actin-sequestering protein to control cytoskeletal reorganization. Here, we investigated whether angiogenesis and tumor metastasis are dependent on hypoxia conditioning-induced TB4 expression in B16F10 melanoma cells. TB4 expression in B16F10 cells was increased by hypoxia conditioning in a time-dependent manner. In addition, we found an increase of angiogenesis and HIF-1α expression in TB4-transgenic (Tg) mice as compared to wildtype mice. When wound healing assay was used to assess in vitro tumor cell migration, hypoxia conditioning for 1 h enhanced B16F10 cell migration. When TB4 expression in B16F10 cells was inhibited by the infection with small hairpin (sh) RNA of TB4 cloned in lentiviral vector, tumor cell migration was retarded. In addition, hypoxia conditioning-induced tumor cell migration was reduced by the infection of lentiviral shRNA of TB4. HIF-1α stabilization and the expression of VEGF isoform 165 and <em>121</em> in hypoxia were also reduced by the infection of lentiviral shRNA of TB4 in B16F10 cells. We also found an increase of tumor <em>growth</em> and lung metastasis count in TB4-Tg mice as compared to wildtype mice. Collectively, hypoxia conditioning induced tumor cell migration by TB4 expression-dependent HIF-1α stabilization. It suggests that TB4 could be a hypoxia responsive regulator to control tumor cell migration in angiogenesis and tumor metastasis.

The mechanism by which the CXC chemokine platelet <em>factor</em> 4 (PF-4) inhibits <em>endothelial</em> cell proliferation is unclear. The heparin-binding domains of PF-4 have been reported to prevent <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> 165 (VEGF(165)) and fibroblast <em>growth</em> <em>factor</em> 2 (FGF2) from interacting with their receptors. However, other studies have suggested that PF-4 acts via heparin-binding independent interactions. Here, we compared the effects of PF-4 on the signalling events involved in the proliferation induced by VEGF(165), which binds heparin, and by VEGF(<em>121</em>), which does not. Activation of the VEGF receptor, KDR, and phospholipase Cgamma (PLCgamma) was unaffected in conditions in which PF-4 inhibited VEGF(<em>121</em>)-induced DNA synthesis. In contrast, VEGF(165)-induced phosphorylation of KDR and PLCgamma was partially inhibited by PF-4. These observations are consistent with PF-4 affecting the binding of VEGF(165), but not that of VEGF(<em>121</em>), to KDR. PF-4 also strongly inhibited the VEGF(165)- and VEGF(<em>121</em>)-induced mitogen-activated protein (MAP) kinase signalling pathways comprising Raf1, MEK1/2 and ERK1/2: for VEGF(165) it interacts directly or upstream from Raf1; for VEGF(<em>121</em>), it acts downstream from PLCgamma. Finally, the mechanism by which PF-4 may inhibit the <em>endothelial</em> cell proliferation induced by both VEGF(<em>121</em>) and VEGF(165), involving disruption of the MAP kinase signalling pathway downstream from KDR did not seem to involve CXCR3B activation.

The effects of prostaglandin E2 (PGE2) and vasoactive intestinal peptide (VIP) on <em>vascular</em> <em>endothelial</em> cell <em>growth</em> <em>factor</em> (VEGF) mRNAs were investigated using lung cancer cells. By RT-PCR, VEGF(<em>121</em>), VEGF(165), and VEGF(189), but not VEGF(206) isoforms were detected in all lung cancer cell lines and biopsy specimens examined. By Northern blot, VEGF mRNA was detected in all small cell lung cancer (SCLC) and non-SCLC (NSCLC) cell lines examined. PGE2, VIP and forskolin caused increased VEGF expression in a time- and concentration-dependent manner using NSCLC cell line NCI-H157. Approximately 1 microM PGE2, 0.1 microM VIP and 50 microM forskolin caused cAMP elevation, 64-, 33- and 128-fold, respectively, using NCI-H157 cells after 5 min. The increase in cAMP caused by PGE(2) and VIP was reversed by somatostatin (SST). Also 1 microM PGE2, 0.1 microM VIP and 50 microM forskolin increased the VEGF mRNA 2.0-, 1.5- and 2.3-fold, respectively, after 4 h. The increase in VEGF mRNA caused by PGE2, VIP and forskolin was inhibited by H-89, a protein kinase A inhibitor. A VIP receptor antagonist, VIPhybrid, inhibited the increase in cAMP and VEGF mRNA caused by VIP. By ELISA, VEGF was detected in the conditioned media exposed to the lung cancer cell lines. These results suggest that VEGF synthesis in and secretion from lung cancer cells can be regulated by agents, which cause adenylyl cyclase activation.

We describe the design and detailed characterization of 6-hydroxy-nicotine (6HNic)-adjustable transgene expression (NICE) systems engineered for lentiviral transduction and in vivo modulation of angiogenic responses. Arthrobacter nicotinovorans pAO1 encodes a unique catabolic machinery on its plasmid pAO1, which enables this Gram-positive soil bacterium to use the tobacco alkaloid nicotine as the exclusive carbon source. The 6HNic-responsive repressor-operator (HdnoR-O(NIC)) interaction, controlling 6HNic oxidase production in A.nicotinovorans pAO1, was engineered for generic 6HNic-adjustable transgene expression in mammalian cells. HdnoR fused to different transactivation domains retained its O(NIC)-binding capacity in mammalian cells and reversibly adjusted transgene transcription from chimeric O(NIC)-containing promoters (P(NIC); O(NIC) fused to a minimal eukaryotic promoter [P(min)]) in a 6HNic-responsive manner. The combination of transactivators containing various transactivation domains with promoters differing in the number of operator modules as well as in their relative inter-O(NIC) and/or O(NIC)-P(min) spacing revealed steric constraints influencing overall NICE regulation performance in mammalian cells. Mice implanted with microencapsulated cells engineered for NICE-controlled expression of the human glycoprotein secreted placental alkaline phosphatase (SEAP) showed high SEAP serum levels in the absence of regulating 6HNic. 6HNic was unable to modulate SEAP expression, suggesting that this nicotine derivative exhibits control-incompatible pharmacokinetics in mice. However, chicken embryos transduced with HIV-1-derived self-inactivating lentiviral particles transgenic for NICE-adjustable expression of the human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF<em>121</em>) showed graded 6HNic response following administration of different 6HNic concentrations. Owing to the clinically inert and highly water-soluble compound 6HNic, NICE-adjustable transgene control systems may become a welcome alternative to available drug-responsive homologs in basic research, therapeutic cell engineering and biopharmaceutical manufacturing.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and thymidine phosphorylase (TP) are important angiogenic enzymes, inducing new blood vessel formation in many human malignancies. In this study, the immunohistochemical expression of the two molecules was analysed in a series of <em>121</em> endometrial carcinomas. VEGF was expressed exclusively in cancer cells, while TP expression was shown in cancer cells (TPcc) and in stromal cells (TPsc) of both fibroblastic and myometrial origin. In all cases, enzymatic detection was particularly evident at the invading tumour front. At this site, TPsc, but not VEGF, expression was associated with non-endometrioid-type carcinomas, high tumour grade, deep myometrial invasion, and advanced stage. VEGF, but not TP, expression was related to increased angiogenesis (p=0.01). Double stratification of the two <em>factors</em>, however, marked VEGF/TPsc co-expression as the most potent angiogenic phenotype (p=0.008), suggesting a synergistic function. Survival analysis revealed that VEGF and TPsc, whether expressed alone or in combination, define poor prognosis. In multivariate analysis, however, stage of disease (p<0.0001, t-ratio 4.4) and VEGF expression (p=0.01, t-ratio 2.4) were the most important prognostic variables. Furthermore, VEGF expression emerged as the only independent prognostic variable in stage I endometrial carcinomas (p=0.04, t-ratio 1.9). This was not shown for TP, probably because of its close association with histopathological parameters. In conclusion, VEGF is a major angiogenic <em>factor</em> in endometrial carcinomas and an independent prognostic <em>factor</em> in stage I endometrial disease. TP is not an effective contributor to the angiogenic process, but is associated with aggressive histological features. The two <em>factors</em>, when co-expressed, play a co-operative role in the induction of angiogenesis.

Altered angiogenic response is associated with high-grade cervical dysplasia and with invasive squamous carcinoma of the cervix. <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> (VEGF) is one of the most potent inducers of angiogenesis and is up-regulated in carcinoma of the cervix. Infection by high-risk human papillomavirus and persistent expression of viral oncogene E6 are etiologically linked to the development of cervical cancer. E6 is able to immortalize cells and induce malignant transformation by inactivating p53. In cervical cancer, regulation of VEGF expression is poorly described. Thus, we investigated whether E6 oncoprotein could regulate VEGF expression in HPV18-positive cervical cancer-derived HeLa cells harboring a wild-type p53. The alternative splicing of vegf mRNA renders three major isoforms of <em>121</em>, 165 and 189 amino-acids in humans. We have designed isoform specific real time QRT-PCR assays to quantitate vegf transcripts and VEGF<em>121</em> was the predominant isoform. Silencing HPV18 E6 mRNA with specific siRNA reduced VEGF<em>121</em> expression by at least 50% whereas silencing of p53 did not alter its expression. Treatment with cycloheximide did not inhibit E6-induced VEGF<em>121</em> expression. Collectively, these results suggest that HPV18 E6 oncoprotein contributes to tumor angiogenesis by inducing VEGF transcription from the promoter in a p53-independent manner.

OBJECTIVE

Our previous work demonstrated that angiogenesis is inhibited in nonhealing venous ulcers. The object of this study was to determine whether local expression of vascular endothelial growth factor (VEGF) and other major regulators of vessel growth are related to healing of venous ulcers.

METHODS

The study included 35 patients with venous ulcers (CEAP 6) and 9 patients whose ulcers had healed (CEAP 5). Control subjects were 18 patients undergoing routine operations (8 with closed suction drains, 10 standard skin biopsies). Healing ulcers were defined as having healed in less than a year from entry to the study; nonhealing ulcers failed to heal in this period. A 1-cm square biopsy specimen was taken from the edge of the ulcer or from a site of lipodermatosclerosis around a healed ulcer. Wound fluids were aspirated from beneath transparent occlusive dressings. Concentrations of VEGF(165) and VEGF-R1 were measured in tissue homogenates with enzyme-linked immunosorbent assay, and results are expressed as mean +/- SEM per milligram of soluble protein (SP). Expression of mRNA transcripts for the VEGF splice variants VEGF(121), VEGF(189), and VEGF(165); the receptors VEGF-R1 and VEGF-R2; the angiopoietins Ang-1 and Ang-2; and their receptor, Tie-2, were measured in biopsy samples with multiplex polymerase chain reaction. Expression of each transcript was normalized to that of the housekeeping gene, GAPDH. Results were analyzed with analysis of variance, t test, and chi(2) test.

RESULTS

There was no difference in VEGF(165) protein concentration between biopsy specimens from healing ulcers (2.12 +/- 0.34 ng/mg SP; n = 18) and nonhealing ulcers (2.36 +/- 0.39 ng/mg SP; n = 12), but concentration was higher in all ulcer samples compared with healthy skin (0.57 +/- 0.20 ng/mg SP; n = 10; P <.01)) and healed ulcers (0.33 +/- 0.06 ng/mg SP; n = 9; P <.01). Concentration of VEGF(165) protein in wound fluid was significantly higher in nonhealing venous ulcers (67.17 +/- 13.87 ng/mg SP; n = 13) compared with healing venous ulcers (32.19 +/- 7.90 ng/mg SP; n = 19; P <.05) or acute wounds (12.26 +/- 4.50; n = 8; P <.01). Concentration of VEGF-R1 was similar in wound fluid obtained from healing ulcers (7.18 +/- 1.34 ng/mg SP; n = 13) and nonhealing ulcers (7.02 +/- 1.21 ng/mg SP; n = 19), and acute wounds (7.12 +/- 2.35 ng/mg SP; n = 8). There was a weak but significant correlation between VEGF(165) protein concentration in the ulcer biopsy specimen and wound fluid from the same ulcer (R(2) = 0.2; P =.019; n = 27). Expression of mRNA for VEGF receptors and Tie-2 was poor. VEGF(121) was expressed in all samples, and VEGF(165) in 43 of 48 samples. mRNA expression of VEGF(189) (P =.001), Ang-1 (P =.002), and Ang-2 (P =.026) was found in more samples from unhealed ulcers than from other sites. Healed ulcers had reduced mRNA expression of VEGF(165) (0.181 +/- 0.003) than did healing ulcers (0.307 +/- 0.016; P =.007) or nonhealing ulcers (0.375 +/- 0.033; P =.001). Relative expression of VEGF(165) to Ang-2 was much lower in healed ulcers (0.4236 +/- 0.060) than in healing ulcers (1.382 +/- 0.235; P =.010) and nonhealing ulcers (1.887 +/- 0.280; P =.003).

CONCLUSIONS

In nonhealing venous ulcers there is a consistently high level of expression of VEGF, at both the gene transcript and protein level. As our previous data demonstrated that angiogenesis is depressed in these poorly healing ulcers, an increase in VEGF production may indicate an increased but ineffectual angiogenic drive. It is also possible that undiscovered inhibitors are released in the ulcer environment.

Uveal melanoma develops in one of the most capillary-rich tissues of the body and is disseminated hematogenously. Knowledge of the nature and the spatiotemporal expression of angiogenic <em>factors</em> in uveal melanoma is essential to the development of new treatment strategies, especially with regard to improving survival. In this study, we measured the angiogenic potential of several angiogenic <em>factors</em> in different uveal melanoma cell lines, in an in vivo model, and in primary tumor material from patients with melanoma. Most uveal melanoma cell lines expressed <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-A (isoforms <em>121</em>, 165, 189), VEGF-B, VEGF-C, VEGF-D, and basic fibroblastic <em>growth</em> <em>factor</em> (b-FGF) to various extents. The expression of VEGF-A <em>121</em> was always higher than that of the other VEGF-A isoforms, suggesting that VEGF-A <em>121</em> is the most abundant VEGF-A isoform. All experimentally induced tumors expressed VEGF-A, VEGF-B, VEGF-C, VEGF-D, and basic fibroblastic <em>growth</em> <em>factor</em> (b-FGF). Similarly, significant amounts of mRNA for VEGF-B, VEGF-C, VEGF-D, and b-FGF were detected in uveal melanoma material from patients. In contrast, VEGF-A mRNA (<em>121</em>, 165, 189) was low (9/28) or not detectable in the tumor samples. The synthesis of VEGF-A 165 and b-FGF protein by various cell lines was measured by enzyme-linked immunosorbent assay (ELISA). Most uveal melanoma cell lines, but not normal melanocytes, strongly synthesized and secreted VEGF-A 165 and b-FGF during cell culture. Our data suggest that the expression of (lymph) angiogenic <em>factors</em> may play a causal role in the angiogenesis and progression of uveal melanoma and distant metastasis.

Infectious spleen and kidney necrosis virus (ISKNV) causes a pandemic and serious disease in fish. Infection by ISKNV causes epidermal lesions, in which petechial hemorrhages and abdominal edema are prominent features. ISKNV ORF48R contains a domain similar to that of the platelet-derived <em>growth</em> <em>factor</em> and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) families of proteins. ISKNV ORF48R showed higher similarity to the VEGFs encoded by Megalocytivirus and Parapoxvirus than to those encoded in fish and mammals. We used zebrafish as a model and constructed a recombinant plasmid containing the DNA sequence of ISKNV ORF48R to study ISKNV infection. The plasmid was microinjected into zebrafish embryos at the one-cell stage. Overexpression of the ISKNV ORF48R gene results in pericardial edema and dilation at the tail region of zebrafish embryos, suggesting that ISKNV ORF48R induces <em>vascular</em> permeability. ISKNV ORF48R is also able to stimulate a striking expression of flk1 in the zebrafish dorsal aorta and the axial vein. Furthermore, ISKNV ORF48R, while cooperating with zebrafish VEGF(<em>121</em>), can stimulate more striking expression of flk1 than can either ISKNV ORF48R or zebrafish VEGF(<em>121</em>) alone. However, decreased expression of FLK-1 by gene knockdown results in the disappearance of pericardial edema and dilation at the tail region of zebrafish embryos induced by overexpression of ISKNV ORF48R in the early stages of embryonic development.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a potent <em>endothelial</em> cell mitogen and angiogenic <em>growth</em> <em>factor</em> that enhances <em>endothelial</em> cell invasion through the extracellular matrix (ECM). While various cell types express VEGF receptors, little is known about the biological actions of VEGF on non<em>endothelial</em> cells. Therefore, the main objective of the present study was to determine the effect of VEGF on the in vitro invasiveness and proliferation of human MDA-MB-231 breast carcinoma cells and human HTR-8/SVneo trophoblast cells. Reverse-transcriptase polymerase chain reaction analysis demonstrated the presence of transcripts encoding VEGF receptors (VEGFR) -1, -2, and -3 as well as neuropilins-1 and -2 in the trophoblast cells, and the presence of transcripts encoding VEGFR-2 and neuropilins-1 and -2 in the breast carcinoma cells. Both cell lines also expressed transcripts for VEGF-A, -B, -C and -D, as well as for placenta <em>growth</em> <em>factor</em> (PlGF). Although incubation with exogenous VEGF-A(165) or VEGF-A(<em>121</em>) did not affect the rate of proliferation of either the trophoblast or the breast carcinoma cells, incubation with these molecules reduced their ability to invade through reconstituted ECM (Matrigel). The effect of VEGF-A(165) on the invasiveness of both cell lines was inhibited by the inclusion of a neutralizing antibody to VEGF. Exogenous VEGF-A(165) also decreased the cell surface expression of the urokinase-type plasminogen activator (a molecule required for invasion) by the breast carcinoma and trophoblast cells. These results indicate that the biological actions of VEGF on certain cell types may differ from the effects of this molecule on <em>vascular</em> <em>endothelial</em> cells, and therefore are relevant to angiogenesis-based therapies.

BACKGROUND

Because vascular endothelial growth factor (VEGF) signalling is deregulated in diabetic retinopathy, the potential therapeutic effects of VEGF inhibitors such as the human VEGF-specific antibody ranibizumab are currently being tested. A study was undertaken to determine whether VEGF-stimulated processes in retinal endothelial cells are reversed by ranibizumab.

METHODS

The influence of VEGF(121) and VEGF(165) on the proliferation and migration of immortalised bovine retinal endothelial cells (iBREC) was studied in the presence and absence of ranibizumab. In addition, the protein composition of tight junctions in the presence of VEGF and its inhibitor in iBREC was investigated.

RESULTS

While both isoforms stimulated proliferation of iBREC, only VEGF(165) influenced cell migration. The addition of ranibizumab counteracted this stimulation without inhibition of the basal levels of migration and proliferation. Plasma membrane staining of the tight junction proteins occludin and claudin-1 disappeared in the presence of VEGF(165); there was no effect on claudin-5 and ZO-1 was only weakly affected. The addition of ranibizumab restored plasma membrane localisation of occludin and claudin-1. For claudin-1, the variation in total protein expression corresponded with the observed effects of VEGF(165) and ranibizumab.

CONCLUSIONS

Ranibizumab reverses proliferation and cell migration stimulated by VEGF and delocalisation of tight junction proteins induced by VEGF(165) in iBREC.

To better understand the poorly <em>vascular</em>ized background of the stroma of pleomorphic adenomas, we attempted to determine the expression of molecules related to blood vessels and hypoxic conditions in pleomorphic adenoma. Surgical specimens and tumor cells in primary culture of salivary pleomorphic adenomas were used for immunohistochemistry for CD31, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and its receptors Flk-1 and Flt-1, as well as for hypoxia markers, such as hypoxia-inducible <em>factor</em>-1alpha (HIF-1alpha) and lactate dehydrogenase-1 (LDH). At the same time, alternative splicing modes of the VEGF gene and expression levels of the HIF-1alpha gene were analyzed in surgical specimens by means of reverse-transcription polymerase chain reaction (RT-PCR) and direct sequencing of the PCR products. In addition to co-immunolocalization with CD31+ <em>vascular</em> <em>endothelial</em> cells, VEGF and its receptors were demonstrated in normal duct epithelial and myoepithelial cells as well as in tumor cells in ductal structures and in myxochondroid stromata. Immunolocalizations for HIF-1alpha and LDH were confirmed in the VEGF-positive area. Immunofluorescence signals for VEGF and others were confirmed in pleomorphic adenoma cells in culture. RT-PCR results showed that there were at least four splicing modes of the VEGF gene, among which VEGF(<em>121</em>) was most enhanced, and higher HIF-1alpha levels in pleomorphic adenomas. The results suggest that pleomorphic adenoma cells produce VEGF in several functional forms for their own proliferation or differentiation, and that the VEGF expression is controlled by hypoxic circumstances of poorly <em>vascular</em>ized pleomorphic adenomas.

Angiogenesis is crucial for tumor <em>growth</em> and dissemination. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a potent angiogenic <em>factor</em> that promotes <em>endothelial</em> cell proliferation and chemotaxis. VEGF occurs as 5 isoforms, as a result of an alternatively spliced transcript that originates from one gene, of which the 2 majors are the VEGF <em>121</em> and 165 isoforms. Our aim was firstly to determine the role of Insulin-like <em>Growth</em> <em>Factor</em>-I (IGF-I) in the regulation of VEGF expression in endometrial adenocarcinoma cells and then the mechanism by which this regulation occurs. IGF-I treatment of HEC-1A cells provoked an increase of VEGF mRNA expression that peaked at 48 hr with a 165 isoform mRNA more abundant than the <em>121</em> isoform. The IGF-I action was confirmed at the protein level, whose concentration was increased in the conditioned media. In experiments using transient transfection of VEGF promoter-luciferase constructs, the IGF-I failed to increase the activity of the VEGF promoter after a 24-hr period of IGF-I treatment, while the addition of Actinomycin D showed an increase of the VEGF mRNA half-life. Most interestingly, Northern blot analysis showed a different stability of the 2 major VEGF isoform mRNAs (VEGF <em>121</em> and 165), of which the <em>121</em> isoform was more stable than the 165 isoform. The IGF-I treatment prolonged the half-life of both of the VEGF isoform mRNAs. Our results suggest that IGF-I regulates VEGF expression in endometrial adenocarcinoma cells at the post-transcriptional level by enhancing the stabilization of the 2 major VEGF isoform mRNAs (VEGF(<em>121</em>) and VEGF(165)). In addition to its proliferative functions, IGF-I induces VEGF expression and participates in the maintenance of an angiogenic phenotype.